Purification of nuclei and preparation of nuclear envelopes from skeletal muscle.
The nuclear envelope is a complex membrane-protein system that is notoriously difficult to purify because it has many connections to both nuclear and cytoplasmic components. This difficulty is compounded by the fact that the nature of these connections vary in different cell types, and so methods must be significantly adapted according to the cell type from which nuclear envelopes are being purified. Here we present a detailed method for purification of nuclear envelopes from one of the most intransigent tissues: skeletal muscle. We further note in the procedure how this method differs from that for other tissues. Identification of nuclear envelope-specific proteins is principally encumbered by endoplasmic reticulum contamination; therefore, we also present a method to purify sarcoplasmic reticulum from muscle.
Wilkie
Gavin S
GS
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
Schirmer
Eric C
EC
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D008967:Molecular Biology / Q000379:methods; D018482:Muscle, Skeletal / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D034881:Nuclear Lamina / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002843:Chromatin; D005456:Fluorescent Dyes